I. Quantitative determination of the amount of DNA per nucleus by interference microscopy

A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment. The values found are slightly low, because, as indicated by biochemical determinations, consistently 5% to 8% of DNA is not extracted by DNase under these conditions. The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7.7 and 15.6 pg (10^-12 g), respectively. Definite advantages of this procedure are: i) comparisons with biochemical determinations to give DNA values for each class of ploidy, ii) comparisons with histophotometry of the Feulgen dye-DNA complex to give absolute values instead of arbitrary units.     

A quantitative determination of the relative amount of histones in polyacrylamide gel by silver stain

On the basis of scanning densitometry of the stained gel, the conditions for the quantitative determination of individual histones by silver was examined and compared with the dye-staining method, in terms of higher sensitivity and faithful quantitation. Fixation with formaldehyde, coupled with simultaneous prestaining with Coomassie brilliant blue (CBB), was found to be most suitable. Prior fixation in acidic alcohol alone failed to stain the histones accurately, but this failure could be partly alleviated by prestaining with CBB. Although the sensitivity for detecting histones by silver staining is lower than that for neutral proteins by about 10-fold, it is at least 10-fold higher than the CBB stain.     

A quantitative study of histones of meiocytes

The existence of a meiocyte-specific histone fraction (FM) (Sheridan and Stern, 1967) is confirmed. FM appears during premeiotic interphase and prophase I in Lilium candidum in addition to a set of standard somatic fractions. Several pieces of evidence were obtained showing that FM can not be an aggregate of non-histone protein with some histone fraction. If the amount of somatic fractions in the total histone is taken as 100% then the relative content of any somatic fraction remains constant (with insignificant fluctuations around the constant level) during the whole period of the rise and fall of FM. Thus, FM appears not at the expence but in excess of somatic fractions, i.e. FM is an extra histone and the resulting histone content of the meiocytes in pachytene is higher than that of somatic and sporogenous cells during mitosis. — The relative amount of FM increase during prophase I reaches a maximum of about 14% over the sum of somatic histone fractions at zygotene-pachytene, and falls to 7% at meiotic divisions. These events coincide with the rise and fall of the synaptonemal complex, and it is therefore the suggested that FM is one of the molecular components of a synaptonemal complex.Dedicated to Professor A. A. Prokofieva-Belgovskaia on the occasion of the seventieth anniversary of her birthday.     

A quantitative study of spermatogenesis in the developing rat testis

Quantitative (stereological) studies were performed to determine the number of germ cells in the developing rat testis. Sprague-Dawley rats aged 1-70 days were fixed by immersion or perfusion and embedded in Epon Araldite. Blocks of tissue were sectioned at 1.5 microns and stained with toluidine blue dye. Sections were systematically scanned and the areal density of nuclear profiles counted using an unbiased counting frame. Numerical density and absolute number of germ cells in the processed block were then estimated. Corrections for processing shrinkage were determined by comparing the volume of processed and unprocessed samples. The results demonstrate the necessity of determining absolute number rather than volume density (or areal density) in comparing germ cell numbers. In these experiments, spermatogonial numbers stabilized in the range 18.4-23.6 million per testis on Day 30. The number of primary spermatocytes that were first apparent on Day 15 increased rapidly to 54.6 million per testis on Day 30 and then slowly to 73.6 million on Day 70. Round spermatids were first apparent on Day 25 and increased rapidly to 85.7 million per testis on Day 40, then continued to increase to 151.9 million on Day 70. The study provides both methods and baseline data for future experiments involving manipulation of the spermatogenic potential of the testis.     

High energy radiation effects in single histones. I. Preparation of histones and irradiation of histone H2B

Histone H2B from calf thymus was irradiated with 50 or 100 ns pulses of 16 MeV electrons in N2O-saturated aqueous solution at pH 9 in the presence of NaN3. All tyrosine moieties in the histone were found to be freely accessible to the attack of .N3 radicals (formed by the reaction .OH + N3(-)----OH- + .N3). At sufficiently high concentrations of H2B, tyrosyl radicals were formed with G(TyrO.) = 5.4/100 eV and dityrosine groups with G(dityr) = 1.6/100 eV, indicating that about 60 per cent of tyrosyl radicals formed bisphenolic products. There is no polymer effect with respect to G(dityr) as inferred from comparison with other authors' data obtained with low molecular weight compounds. Kinetic measurements revealed that tyrosyl radicals reacted in two modes, a fast one with a value of tau 1/2 of about several milliseconds and a slow second order process also in the millisecond range. The fast process is assigned to intramolecular reactions of tyrosyl radicals generated in close proximity to each other and the slow process to intermolecular self reactions of isolated tyrosyl radicals distributed statistically in the solution. There is a polymer effect with respect to the rate constant of the slow process: 2k8 = 4.8 X 10(7) dm3 mol-1 s-1 (H2B) and 2k8 = 4 X 10(8) dm3 mol-1 s-1 (Lys-Tyr-Lys, Prütz et al. (1983)). The five histones contained in calf thymus were isolated chromatographically with the aid of two gels, Bio-Gel P-60 (BioRad) and Sephadex G100 (Pharmacia).     

Amino acid composition of sperm histones in the house cricket Acheta domesticus.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.     

Detection of mucus glycoconjugates in human cervical epithelium by lectin-colloidal gold technique in transmission electron microscopy. I. A quantitative study during the menstrual cycle

We investigated the presence and distribution of five glycosidic residues at the level of the endocervical columnar epithelial cells during the various phases of the menstrual cycle in healthy subjects. We utilized the lectins Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Soybean Agglutinin (SBA), Concanavalin A (ConA) conjugated with colloidal gold particles as ultrastructural markers in Transmission Electron Microscopy. This technique proved to be particularly suitable for detecting glycosidic residues at the cellular level, where the mucus is produced. All the lectin-binding sites (except ConA binding sites which were not detected) appeared to be produced in significantly different amount during the cycle, supporting the hypothesis that the cyclic-related changes of the cervical mucus viscoelasticity is due to glycoconjugate cyclic variations.     

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.     

A study of histones in amoeba nuclei by indirect immunofluorescence method.

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

A qualitative and quantitative study of subfractions of the histone H10 in various mammalian tissues.

The histone H10 was examined from seven mammalian species. All tissues were shown to contain two subfractions of H10, except for those of rabbit, in which little or no H10 was found. The subfraction composition was compared quantitatively in different mouse and hamster tissues, with the conclusion that this composition is tissue-specific. It is proposed that the wide occurrence of H10, together with the evidence of no more or less than two subfractions wherever it occurs, and the tissue-specific nature of the ratio of subfractions, signify that these two subfractions have specific individual functions.     

Chemical cross-linking of H1 histone to the nucleosomal histones.

When whole steer kidney nuclei were treated with dimethyl-3,3'-dithiobisproprionimidate, N,N'-bis(2-carboxyimidomethyl) tartaramide dimethyl ester, or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under approximately physiological ionic conditions, H1 histone was cross-linked to each of the four histones in the nucleosome core. The carbodiimide reagent, which introduces no atoms between the amino acid side chains being joined, seemed to give the same result as did the longer di-imidate cross-linking reagents. When conditions were optimized for the production of of H1-containing dimers, the total yield of H1-core histone heterodimers was nearly equal to the yield of H1 homodimers. Naturally occurring H1 dimers and cross-linked heterodimers of high mobility group proteins 14 and 17 with H1 and core histones were also observed.     

Antisera to individual histone fractions of the calf thymus. II. A study of the immunochemical specificity of the histones by an indirect immunofluorescence method]

Immune antisera to 5 fractions (H1, H2a, H2b, H3, H4) of calf thymus histone were assayed using indirect immunofluorescence (IIF). The analysis of such sera by this technique, as well as the data on complement fixation obtained previously, show that these antisera are highly active and specific for various test-objects: thymys, liver nuclei of rat, chicken, and calf, chicken erythrocytes, metaphase chromosomes of mouse fibroblasts. These antisera are of importance for the evaluation of species- and tissue-specificity of different histone fractions. Using the IIF reaction, a comparison was made between the nucleosome fraction H3, which is evolutionary stable, and fraction HI from calf thymus, rat and chicken liver, and chicken erythrocytes.